Glycated proteins are generated by non-enzymatic covalent bonding between aldehyde groups in aldoses, such as glucose (monosaccharides potentially containing aldehyde groups and derivatives thereof), and amino groups in proteins, followed by Amadori rearrangement. Examples of amino groups in proteins include α-amino groups of the amino terminus and side chain ε-amino groups of the lysine residue in proteins. Examples of known glycated proteins generated in vivo include glycated hemoglobin resulting from glycation of hemoglobin and glycated albumin resulting from glycation of albumin in the blood.
Among such glycated proteins generated in vivo, hemoglobin A1c (HbA1c) has drawn attention as a glycemic control marker significant for diagnosis of diabetic patients and control of conditions in the field of clinical diagnosis of diabetes mellitus. HbA1c is a protein comprising glucose bound to the α-amino group at the N-terminal (amino-terminal) valine (Val) residue of the hemoglobin “β chain.” The blood HbA1c level reflects the average blood glucose level for a given period of time in the past, and the measured value thereof serves as a significant indicator for diagnosis and control of diabetes conditions.
Several types of enzymatic methods involving the use of amadoriases have heretofore been known as methods for rapidly and simply measuring HbA1c.
Enzymes that oxidize iminodiacetic acid or a derivative thereof (also referred to as an “Amadori compound”) in the presence of oxygen to catalyze a reaction to generate glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide are collectively referred to as “amadoriases.” Amadoriases are known to be useful for measuring HbA1c by an enzymatic method. An example of a substrate that is known to be oxidized by amadoriases is α-fructosyl valyl histidine (hereafter referred to as “αFVH”).
Amadoriases have been found in bacteria, yeast, and fungi. For example, amadoriases derived from the genera Coniochaeta, Eupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Aspergillus, Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella, Achaetomium, Thielavia, Chaetomium, Gelasinospora, Microascus, Leptosphaeria, Ophiobolus, Pleospora, Coniochaelidium, Pichia, Debaryomyces, Corynehacterium, Agrobacterium, and Arthrobacter have been reported (e.g., Patent Documents 1 and 6 to 15 and Non-Patent Documents 1 to 9). These genera may be referred to as the genera Coniochaeta etc. in this description. In some of the aforementioned documents, an amadoriase may also be referred to as, for example, ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, or fructosyl amine oxidase. These terms are synonymously used herein.
As a method for rapidly and readily measuring HbA1c with the use of various types of amadoriases as described above, a method in which HbA1c is degraded with a cleavage enzyme such as a protease or the like, and a particular target substance released from the β-chain amino terminus of HbA1c is quantified with the use of amadoriases as described above is known (e.g., Patent Documents 1 to 7).
Specifically, a method in which HbA1c is degraded with a particular protease or the like, αFVH is released from the β-chain amino terminus thereof, and the released αFVH is quantified has been known. At present, such method is a major technique for measuring HbA1c by an enzymatic method.
However, in order to sufficiently hydrolyze the HbA1c β chain down to αFVH within a short period of time, it is necessary to formulate the assay reagent with large quantities of proteases and/or many different types of proteases exhibiting different types of reactivity. However, such inclusion is not preferable for the reasons described below.
That is, a protease is capable of protein hydrolysis and, therefore, enzymes which are proteins are also hydrolyzed by a protease. As such, amadoriases will also be hydrolized by a protease and inactivated, and, as a result, the reaction consuming a glycated peptide and oxygen to generate hydrogen peroxide will be inhibited. In order to address such problem, it is possible to increase the amount of amadoriases, and to complete the measurement before amadoriases are completely inactivated. However, increasing the amount of amadoriases will lead to preferential hydrolysis of amadoriases rather than HbA1c, which intrinsically is not preferable.
When measuring HbA1c by allowing an amadoriase to react with αFVH and quantifying the resulting hydrogen peroxide, hydrogen peroxide may be quantified using a peroxidase. In such a case, the peroxidase will also be hydrolyzed by the protease and be inactivated, which is not preferable.
As another aspect, when measuring HbA1c using enzymes, it is commonplace to use an automated analyzer. In such a case, a single sample is simultaneously subjected to analysis of various biomarkers including HbA1c. Since each biomarker is analyzed using an enzyme or antibody, upon contamination of a protease, the enzyme or antibody contained in the biomarker reagent may be hydrolyzed. In such a case, biomarkers other than HbA1c may not be accurately analyzed. Accordingly, it is preferable not to include a protease in a reagent to be mounted on an automated analyzer.
For the reasons described above, it was desired to minimize the amount and the types of proteases to be applied in the method comprising degrading HbA1c with a particular type of protease or the like, releasing αFVH from the β chain amino terminus thereof, and quantifying the released αFVH. If the amount and the types of proteases to be applied are minimized, however, the HbA1c β chain may not be sufficiently hydrolyzed down to αFVH, and, as a result, a wide variety of glycated peptides generated during the process of hydrolysis would also be present.
In addition, the speed of the hydrolysis reaction by a protease depends on the substrate concentration. Therefore, if the majority of HbA1c is hydrolyzed, then the protease reaction speed will be lowered significantly. Accordingly, at the time of completion of measurement of HbA1c, i.e., quantification of αFVH, it is deduced that although the majority of HbA1c is hydrolyzed down to αFVH, glycated peptides that are not hydrolyzed down to αFVH also remain.
As such, if remaining glycated peptides derived from the HbA1c β chain that were not hydrolyzed down to αFVH can be simultaneously quantified, then the amount of proteases to be applied can be reduced, and/or the sensitivity of the method for measurement of HbA1c using an amadoriase can be enhanced. However, no amadoriases exhibiting enzymatic activity on a wide variety of glycated peptides have been found in the past.